Aim. Orthopedic implants play a tremendous role in fixing bone damages due to aging as well as fractures. However, these implants tend to get colonized by bacteria on the surface, leading to infections and subsequently prevention of healing and osteointegration. Recently, Roupie et al. showed that a nisin layer-by-layer based coating applied on biomaterials has both osteogenic and antibacterial properties. The Galleria mellonella larva is a well-known insect infection model that has been used to test the virulence of bacterial and fungal strains as well as for the high throughput screening of antimicrobial compounds against infections. Recently, we have developed an insect infection model with G. mellonella larvae to study implant-associated biofilm infections using Kirschner (K)-wires as implant material. Here, we would like to test the antibacterial capacity of nisin layer-by-layer based coatings on K-wires against Staphylococcus aureus in the G. mellonella larva implant infection model. Method. Prior to the implantation procedure, G. mellonella larvae are maintained at room temperature on wheat germ in an incubator. The larvae received bare titanium K-wires (uncoated), or either control-coated or nisin-coated K-wires. After one hour, the larvae were injected with 5×10. 5. S. aureus bacteria per larva (i.e., hematogenous implant infection model). Next, the larvae were incubated at 37. o. C in an incubator and the survival of the larvae was monitored for five days. Moreover, the number of bacteria on the implant surface and in the surrounding tissue was determined after 24h of incubation. Further, scanning electron microscopy (SEM) analyses were performed to study the effect of nisin on biofilm formation. Results. The larvae receiving the nisin-coated K-wires showed significantly higher survival rates compared to uncoated titanium K-wires, although not when compared to control-coated K-wires. A more than 1-log reduction in number of bacteria on the implant surface and in the surrounding tissue was observed in larvae receiving the nisin-coated K-wires, when compared to uncoated titanium K-wires SEM analysis showed reduced colonization of the bacteria nisin-coated K-wires compared to the controls. Conclusions. In conclusion, the antimicrobial nisin layer-by-layer based coating applied on titanium surfaces is able to prevent implant-related S. aureus biofilm infection in G. mellonella and is a promising antimicrobial strategy to prevent implant-related infections

Background. Two-stage revision arthroplasty is the standard treatment for chronic hip and knee periprosthetic joint infections (PJI). Accurate diagnosis of persistent infections at 2nd stage using established biomarkers and diagnostic criteria is of paramount importance. This study aimed to evaluate the diagnostic value of synovial calprotectin and alpha-defensin, and compare established diagnostic criteria from the International Consensus Meeting (ICM 2018) and the European Bone and Joint Infection Society (EBJIS 2021) to determine persistent PJI at the 2nd stage of a two-stage revision arthroplasty. Methods. We retrospectively analyzed 97 patients who underwent 100 two-stage revisions (hip: 39, knee: 61). Synovial fluid samples were assessed for calprotectin and alpha-defensin levels. ICM 2018 and EBJIS 2021 were applied to all patients undergoing 2nd stage revision. Receiver operating characteristic (ROC) curves and Youden Index were utilized to determine optimal cut-off values, and correlations between biomarkers were evaluated. The microbiological spectrum was analyzed at 2nd stage and re-revision surgery. Results. Calprotectin levels showed a sensitivity of 66.7%, specificity of 32.9%, and accuracy of 38.0% in predicting septic failure. Alpha-defensin showed sensitivity of 28.6%, specificity of 87.8%, and accuracy of 79.2%. Significant correlations included: calprotectin with PMN% (r = 0.471, p = 0.05) and alpha-defensin with WBC (r = 0.830, p < 0.01) in the successful cohort. For septic re-revisions, calprotectin and alpha-defensin were highly correlated (r = 0.969, p < 0.01). ICM correctly diagnosed persistent PJI in 26.7%, while EBJIS diagnosed 24.2%. The microbial spectrum shifted from gram-positive to gram-negative bacteria between reimplantation and re-revision surgeries. Conclusion. Synovial calprotectin and alpha-defensin demonstrated limited accuracy in ruling out persistent PJI at reimplantation. The low sensitivity of current diagnostic criteria, combined with the observed shift in microbial spectrum, underscores the challenges in diagnosing persistent PJI during 2nd stage of a two-stage revisions arthroplasty

Aim. The SOLARIO trial is a randomised controlled non-inferiority trial of antibiotic strategy for bone and joint infection. SOLARIO compares short or long post-operative systemic antibiotic duration, for patients with confirmed infections, who had local antibiotics implanted and no infected metalwork retained when undergoing surgery. This analysis compared systemic antibiotic use in the short (intervention) and long (standard of care) arms of the trial, in the 12 months after index surgery. Method. Data was collected prospectively from study randomisation, within 7 days of index surgery. All systemic antibiotics prescribed for the index infection were recorded, from health records and patient recall, at randomisation, 6 weeks, 3-6 months and 12 months after study entry. Start and end dates for each antibiotic were recorded. Results. 251 patients were randomised to short systemic antibiotics (up to 7 post-operative days) and 249 patients, to long systemic antibiotics. 5 participants in the short group and 2 participants in the long group withdrew from study follow-up. Complete data for all systemic antibiotics taken in the 12 months following surgery, were available for 237 participants in the short group and 236 participants in the long group. 80 participants across both groups were noted as having deviated from their assigned treatment strategy. Both groups received empiric antibiotics, predominantly vancomycin and meropenem, for up to 7 days after surgery. Considering each prescribed antibiotic as a separate duration (even when administered concurrently), participants assigned to standard care received a mean of 74.9 antibiotic-days. Participants assigned to short systemic antibiotics received a mean of 27.5 antibiotic-days in the 12 months after surgery. The most commonly prescribed antibiotics in both treatment groups were vancomycin and meropenem: these antibiotics accounted for 7.1 days prescribed per participant in the long group, and 6.3 days in the short group (p=0.37). Reasons for post-randomisation antibiotic prescribing in the short treatment group included later planned surgery, identification of bacteria requiring additional systemic antibiotics, and treatment of superficial wound infections. WHO AWaRe classification ‘watch’ and ‘reserve’ group antibiotics, such as ciprofloxacin, rifampicin, vancomycin and meropenem, accounted for 39.4 antibiotic-days per long group participant, and 16.5 antibiotic-days per short group participant. Conclusions. Considering the combined duration of all systemic antibiotics prescribed over 12 months, including those co-administered, participants in the short arm of the SOLARIO trial received considerably fewer days of all antibiotic classes, and particularly those antibiotics restricted in the WHO AWaRe classification (2021)

Aim. The management of PJIs is slowed down by the presence of bacteria forming biofilms where they may withstand antibiotic therapy. The use of adjuvant strategies, such as hydrolytic enzymes cocktail targeting biofilm matrices and facilitating their dispersion, is a promising option to limit impact of biofilms. Our aim was to evaluate the effect of enzymes cocktail combined with antibiotic dual therapy of rifampicin and vancomycin in a relevant in-vitro model. Method. Mature methicillin-resistant Staphylococcus aureus biofilms were grown on Ti-6Al-4V coupons by adding 1mL of a 8Log10 ATCC 33591 suspension in TGN (TSB + 1% glucose + 2% NaCl) to 24-wells plates containing the coupons and incubating the plates for 24h at 37°C with a continuous 50rpm agitation. The samples were rinsed and placed in 6 wells plates containing 1ml of the enzymatic cocktail (C.D.D.) solution (tris-buffered (pH 7.0) solution of 400 U/ml of aspecific DNA/RNA endonuclease, 50 U/ml of endo-1,4-b-D-glucanase, and 0.06 U/ml of β-N-acetylhexosaminidase). 9ml of TGN or TGN containing antibiotics RIF/VAN (rifampicin 5µg/mL + vancomycin 8µg/mL) at clinically relevant concentrations found locally in bone or joints, was then added and the samples were incubated in identical conditions for 24h. The samples were then recovered and rinsed. CFU counts were obtained by recovering the bacteria with sonication, serial dilutions, and TSA plating. Biomass was determined via crystal violet staining, followed by dye solubilization in acetic acid, and absorbance measurement using a spectrophotometer. Results. Significant reductions in bacterial counts were observed in biofilms exposed to either RIF/VAN or RIF/VAN+CDD, by respectively 2,6 and 3,7Log10 when compared to samples reincubated with TGN alone (p <0.05). Additionally, CFU counts in samples exposed to RIF/VAN+CDD were reduced by 1,1Log10 when compared to those exposed to RIF/VAN (p<0,05). Significant reduction in biomass (-29,8%, p<0.05) was observed for coupons exposed to RIF/VAN+CDD when compared to C.D.D alone (figure 1). Conclusions. The concurrent utilization of enzymes with rifampicin and vancomycin, holds promise as a feasible method to address periprosthetic joint infections (PJIs). For any tables or figures, please contact the authors directly

Aim. Diagnosis of prosthetic joint infection are often complicated by the presence of biofilm, which hampers bacteria dislodging from the implants, thus affecting sensitivity of cultures. In the last 20 years several studies have evidenced the usefulness of implant sonication to improve microbial recovery from biofilm formed on inert substrates. More recently, treatment of prosthetic joints and tissues with Dithiothreitol, a sulphur compound already used in routine diagnostic workflow for fluidification of respiratory samples, has proved to be not inferior to sonication in microbiological diagnosis of prosthetic joint infections. This study aimed to evaluate if the combination of the two treatments could further improve microbial retrieval from biofilm in an in vitro model. Method. Three isolates of Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus lugdunensis, Eschericha coli and Pseudomonas aeruginosa responsible of prosthetic joint infections were used. They were grown onto 3 titanium discs (20 mm diameter) and incubated in 3 sterile plastic containers with 15 mL of Triptyc Soy Broth. After overnight incubation, not adhered cells were removed and fresh broth was added to each sample. After 48 hours incubation, the exausted broth was removed and one sample was used for sonication, one for treatment with 0,1% (v:v) Dithiothreitol and one treated with Dithiothreitol followed by sonication. Treated fluids were plated on Muller Hinton Agar plates for colony count. One-way ANOVA analysis was performed to evidence statistical differences between treatments. Results. Similar colony counts were observed for the 3 treatments: 10.1± 0.77 log CFU/mL for Dithiothreitol, 10.0 ± 0.75 for sonication and 10.1 ±0.73 for dithiothreitol + sonication. No statistical differences between the 3 treatments were evidenced by ANOVA analysis. Conclusions. Results seems to confirm that treatment with dithiothreitol is equivalent to sonication in recovering bacteria from biofilm grown on inert surface. Combining dithiotreitol treatment with sonication does not significantly improve bacterial recovery in respect to each treatment alone

Aim. The primary objective is to evaluate the diagnostic performance of inoculating homogenized tissue and bone biopsies in blood culture bottles (BCB) for patients with (suspected) orthopaedic device-related infections. As secondary objective the time to positivity (TTP) of BCB and Wilkins-Chalgren broth (conventional method) will be evaluated. Method. Patients undergoing revision surgery due to suspected or proven fracture-related infection (FRI) or periprosthetic joint infection (PJI) according to respectively Consensus definition and EBJIS definition are included. 1,2. A minimal of three macroscopic infected/inflamed tissue/bone samples are collected in a container with saline and glass beads. 1.5 mL of the homogenized suspension is inoculated in BacT/ALERT FA and FN Plus bottles for 14 days. The remaining suspension is inoculated in Wilkins-Chalgren broth for 10 days and subcultured when cloudy or after 10 days. TTP is defined as the time until definite identification of the pathogen in the Laboratory Information System. Results. Up to now, 25 patients have been included, 11 (44%) had concordant results in BCB and the CM. In 11 patients cultures showed negative results for both methods. Three patients tested positive with BCB but remained negative with the same pathogen in CM. In the first patient, the CM failed to identify anaerobic bacteria (i.e. Fusobacterium nucleatum). In the second patient, three BCB were positive with Staphylococcus capitis. The third patient showed an infection with Escherichia coli, which was detected in all samples from the BCB, while all cultures obtained with the CM remained negative. A possible explanation for this discrepancy could be that this patient already received antibiotic therapy. BCB contain resins, which are capable of neutralizing antibiotic activity. Another case illustrating superiority of BCB involved an infection with Cutibacterium acnes, which showed positivity in six BCB, while only three were positive using the CM. We observed the shortest TTP with BCB. The median TTP of BCB was 32.0 hours (IQR 29.8) compared to a median TTP of 77.5 hours (IQR 107.6) when culturing with the CM. Contamination was seen in three patients with both methods, in eight patients contamination was only seen with the CM. For the remaining 14 patients no contamination was found. Conclusions. The results in this ongoing study indicate that the recovery of pathogens and TTP is better using BCB compared to CM. In addition, contamination occurs less frequently with the BCB method. Culturing tissue or bone biopsies in BCB seems a promising and faster detection method

Aim. Biomaterial-associated infections (BAI) present a formidable clinical challenge. Bioactive glasses (BG) have proven highly successful in diverse clinical applications, especially in dentistry and orthopaedics. In this study, we aimed to determine the effect of three commonly used BG composition and particle sizes on cell and bacterial attachment and growth. Our focus is on understanding the changes in pH and osmotic pressure in the surrounding environment during glass degradation. Method. First, three different melt-derived glasses were characterized by analyzing particle size and glass network structure using Raman and NMR. The different glasses were then tested in vitro by seeding 4x 10. 4. cells/well (SaOS Cell line) in a 48 well plate. After a pre-incubation period of 72 hours, the different BGs and particle sizes were added to the cells and the pH value, ion release and live/dead staining was measured every hour. The effect of BG against bacteria (S. epidermidis) was analyzed after 24 and 72 hours of treatment by using XTT viability assay and CFU counting by plating out the treated aliquot agar to estimate the viable bacteria cells. Results. All three BG compositions tested showed a significant increase in pH, which was highest in BG composition 45S5 with a value of 11 compared to the other BG compositions 10 and 9 in S53P4 and 13-93 respectively. This strong increase in the pH in all BG samples tested results in a strongly reduced cell viability rate of more than 75% compared to the untreated control and 6-fold reduction in bacterial viability compared to the untreated control. The live/ dead assay also showed an increased cell viability with increasing glass particle size (i. e smallest glass particle < 25% viable cell and largest glass particle> 65% viable cell). The ion release concentration over 50 h showed an increase in sodium ions to 0.25 mol/L, calcium to 0.003 mol/L and a decrease in phosphorus. Conclusions. These results show that the composition of the bioactive glass and the choice of particle size have a major influence on subsequent applications. In addition to the different compositions of the BG, particle size and additional medium change also influence the pH and ion release, and therefore also on cells or bacteria viability. The sizes of the bioactive glass particle are inversely proportional to it. Further tests are necessary to develop custom design BG compositions, which simultaneously stimulate osteoblasts proliferation and prevent microbial adhesion

Aim. The osteolytic process of osteomyelitis is, according to textbooks, caused by increased osteoclast activity due to RANKL production by osteoblasts. However, recent findings contradict this theory. Therefore, the aim was to investigate, in a porcine osteomyelitis model, how osteolysis is affected by massive inflammation and RANKL blocking, respectively. In parallel, patients with chronic osteomyelitis, diabetes, foot osteomyelitis, and fracture related infections (FRI) were included for advanced histological analysis of osteolysis. Methods. In pigs, a tibial implant cavity was created and inoculated with 10. 4. CFU of Staphylococcus aureus: Group A (n=7). Group B (n=7); + 1cm. 3. spongostan into the cavity. Group C (n=4); + systemic Denosumab treatment. Spongostan was used as an avascular material to support bacterial growth and thus increase the inflammatory response. Denosumab treatment was administrated to suppress osteoclast activity by RANKL inhibition (as in osteoporotic patients). The volume of osteolysis was accessed by CT scans. Immunohistochemistry with antibodies towards Cathepsin K was used to identify osteoclasts within the bone lesions. Briefly, the number of Cathepsin K positive cells, i.e., both precursors and bone resorbing osteoclasts, respectively, were counted in 10 high power fields (400x). In total, 50 bone infection patients were included (Herlev Hospital). From each patient five parried samples were taken for histology and microbiology, respectively. Histopathology, CT osteolysis volume estimation, and molecular expression of osteoclasts and inflammatory markers are ongoing. One FRI patient was osteoporotic and treated with Denosumab for 6 years. Results. All pigs were confirmed infected in the implant cavity. The volume (2.41 ± 1.29cm. 3. ) of osteolysis was significantly increased in the spongostan group in comparison to Group A (1.24 ± 0.59 cm. 3. ) (p=0.04). Thereby, the spongostan group had bacteria deeper into the bone from the inoculation point. Sufficient Denosumab treatment, i.e. reduced serum Ca was seen in 3 pigs. None of the Denosumab treated pigs showed reduced osteolysis in comparison to Group A (1.42 ± 0.63 cm. 3. ). The Cathepsin K score of Group C was 17 (15-23 IQR) of precursor osteoclasts and 2 (0-2 IQR) of osteoclasts in Howship lacunae. The Denosumab treated patient showed substantial osteolysis and histological analysis confirmed acute inflammatory. Conclusions. Application of spongostan, i.e., bacterial host optimization and massive inflammation promotes osteolysis and local bacterial dissemination. Osteoclast blocking with Denosumab showed no impact on osteolysis. Elucidation of the pathophysiology causing bone loss in osteomyelitis is fundamental. However, the widely accepted osteoclast-based theory might not be the only relevant

Aim. Prosthetic joint infections (PJI) remain a great challenge in orthopedic surgery with a high mortality rate. It is particularly complicated by biofilms and infections caused by Methicillin-resistant Staphylococcus aureus (MRSA). It concurrently shields bacteria from host immune responses and confers resistance to antibiotics. This study aims to investigate the efficacy of radioimmunotherapy as an innovative therapeutic modality to address the challenges posed by MRSA and its biofilm. Method. We induced specific monoclonal antibodies 4497-IgG1 as carriers, which target wall teichoic acids (WTA) existing on MRSA and its biofilm. Radionuclides actiniumr-225 (. 225. Ac, α-emitter) and lutetium-177 (. 177. Lu, β-emitter) were conjugated with mAbs using DOTA as chelator. Quality control was assessed using thin layer chromatography and immunoreactivity assays. . 225. Ac- and . 177. Lu-labelled 4497-IgG1 were employed to evaluate the susceptibility of MRSA and its biofilm to the radioimmunotherapy in vitro. Planktonic MRSA and biofilms, at concentrations of 10. 8. and 10. 7. CFU/mL, were incubated at 37°C for 60 minutes in PBS containing either . 225. Ac-mAb (0 - 14.8 kBq) or . 177. Lu-mAb (0 - 14.8 MBq). Radiolabelled dunituximab and free radionuclides serve as isotype-matched negative control. The bacterial viability and metabolic activity were subsequently quantified using CFU and XTT assays. Results. The radiochemical purity of the . 225. Ac-mAbs and . 177. Lu-mAbs complex were determined to be 95.4% and 96.16%. Immunoreactivity fractions of them were measured at 81.8% and 80.8%. . 225. Ac-mAbs and . 177. Lu-mAbs exhibited significant and dose-dependent antimicrobial effects on both planktonic MRSA and biofilm. . 225. Ac- and . 177. Lu-4497IgG1 at doses of 7.4 kBq and 7.4 MBq resulted in more than 4-log reduction in bacterial counts. In biofilms, 2-log reduction at the highest . 225. Ac radioactivity of 14,8kBq. The . 177. Lu complex showed a strong dose-dependent effect, with a reduction of up to 4-log. The XTT assay confirmed these findings, showing a decrease in metabolic activity corresponding to a decrease in bacterial counts, and a slight increase in metabolic activity at the lower dose. Conclusions. Our study demonstrates the efficacy of . 225. Ac and . 177. Lu-labelled 4497-IgG1 antibodies in mediating dose-dependent bactericidal effects against planktonic MRSA and biofilms in vitro. This indicates that radioimmunotherapy could be a potential targeted therapeutic strategy against MRSA and its biofilm. Further research in preclinical and clinical settings is warranted to validate and refine these findings on biofilm-associated implant infections

Aim. Aim of this study was to establish the first clinical results after implantation of ultrathin silver-polysiloxane-coated. 1. plates in the treatment of infected non-union of the femoral shaft. Method. As part of the REFECT study, a prospective, non-interventional analysis was conducted encompassing all patients who received internal stabilization with a silver-coated. 1. plate from 01/2023 to 09/2024 as part of the treatment for infected non-union of the femur. Standardized clinical follow-ups including PROMs (WOMAC-Index, LEF-S, EQ-5D, VAS) and X-rays were performed 3, 6, 12 (and 24) months postoperatively. For comparison, a retrospective analysis of 76 patients with infected femoral non-union, who had received a stabilization with an uncoated plate in the past 10 years, was performed. Results. The mean follow-up of the 8 included patients (mean bone defect: 3.6 cm) was 9 months (as of 04/24). Multiresistant bacteria were found in the intraoperative samples of 5 patients. The concentration of silver ions in blood serum reached a maximum of 0.014 mg/l in the laboratory controls. All patients showed a positive healing process with no sign of re-infection and no adverse procedure-associated events. Full weight bearing was achieved after an average of 4 months (n=6) with improved WOMAC-, LEF-S-, EQ-5D and VAS-score at 1-year FU. In the reference group (uncoated, mean FU: 3.5 years), there was a re-infection rate of 25 %, mostly in the first 2 years. Difficult-to-treat bacteria were detected in 22%, multiresistant Staph. epidermidis in 28% of cases. Conclusions. -. The silver-coated. 1. implants showed good biocompatibility with no evidence of procedure-associated complications. -. The use of silver-coated. 1. implants could reduce the risk of re-infection. -. Further clinical data with longer follow-up are needed to assess the long-term value of the procedure

Aim. To date, no ultimate diagnostic gold standard for prosthetic joint infections (PJI) has been established. In recent years, next generation sequencing (NGS) has emerged as a promising new tool, especially in culture-negative samples. In this prospective study, we performed metagenomic analysis using 16S rRNA V3-V4 amplicon NGS in samples from patients with suspected PJI. Methods. A total of 257 (187 culture-negative (CN) and 70 culture-positive (CP)) prospectively collected tissues and sonication fluid from 32 patients (56 revisions) were included. 16S rRNA V3-V4 amplicons were sequenced using Illumina's MiSeq (California, USA) followed by bioinformatic analysis using nf-core/ampliseq pipeline. Results. We successfully sequenced 255 samples and detected a total of 105 microorganisms. These were mainly environmental microorganisms present in a small number of reads (≤100), indicating possible contamination. Pseudomonas spp. (non-aeruginosa species) was detected most frequently in 73% (187/255) of samples. The test showed limitations in species classification and identified microorganisms mainly at genus level. Significant differences in the number of reads were observed when comparing CN (≤100) and CP (≥1000) samples. In two CP, no bacteria were identified with sequencing, which is probably due to low bacterial load (1 CFU. Haemophilus spp. was detected with a significant number of reads (≥10000) in five samples from a single patient, in whom infection was considered likely according to EBJIS criteria, changing it to confirmed infection. Staphylococcus spp. was identified with ≥10000 reads in two CNs from an individual who was receiving antibiotic treatment at the time, had clinical signs of infection, and had a confirmed infection with S. lugdunensis one month earlier. Cutibacterium spp. with 36% (93/257) and Staphylococcus spp. with 34% (87/257) were detected with a minimal number of reads (≤100) in several CN, indicating possible contamination with normal skin microbiota. In one patient, Facklamia spp., an opportunistic pathogen, was detected in two samples by sequencing, but not by culture. Conclusions. We consider 16S rRNA V3-V4 amplicon sequencing to be a promising tool; however, further studies are needed to clarify uncertainties regarding the interpretation of the results in combination with other criteria. Using this method, we were able to successfully confirm infection in two patients whose microbiological results were initially negative, leading to a change from likely to confirmed infection in one case. The thresholds and interpretation of the results are currently unclear, therefore the method is being used experimentally rather than diagnostically at the time of writing

Aim. Irrigation and debridement with an irrigation solution are essential components of the surgical management of acute and chronic periprosthetic joint infection (PJI). Nevertheless, there is a lack of agreement regarding the most effective solution to use. The aim of the study was to perform a systematic review and meta-analysis of the current literature concerning the efficacy of different irrigation solutions over bacterial biofilm. Method. This study was conducted in accordance with the Preferred Reporting Items for Systematic reviews and Meta-Analysis extension for Network meta-analysis (PRISMA-NMA) checklist for systematic reviews and meta-analyses. A comprehensive literature search of PubMed, Cochrane Library, Web of Science and Scopus databases from inception to September 1, 2023. We combined terms related to PJI, biofilm and irrigation solutions studied in vitro. We performed a network meta-analysis to analyze which irrigation solution achieved a higher reduction of colony forming units (CFU) after specific exposure times, always with a maximum of five minutes, replicating intraoperative conditions. Effect-size was summarized with logarithmic response ratio (logRR) and 95% confidence intervals (95% CI). The rank probability for each treatment was calculated using the p-scores. Results. We screened 233 potential sources. Following deduplication, screening and full-text review, four studies with ten irrigation solutions for different duration of exposures were included, always less than five minutes, replicating intraoperative conditions. Solutions were studied over mature biofilms of most frequent bacteria grown over metal, bone cement or polyethylene surfaces. The highest effect was achieved with povidone iodine 10% during 5 minutes (logRR: −12.02; 95% CI: −14.04, −9.99). The best ranked solutions were povidone iodine 10% during five, three and one minute (respective p-scores: 0.977, 0.932, 0.887) and its combination with hydrogen peroxide for 3 minutes (p-score: 0.836). Povidone iodine 0.3% acting for 5 minutes completed the top 5 best ranked solutions in this study (p-score: 0.761). We assumed that there were no inconsistencies in our network because after examining both scenarios, with and without inconsistencies, the results were not significantly different. Conclusions. Our results show that 10% povidone-iodine is the best antiseptic solution when studied in vitro in the context of prosthetic joint infection. However, the included studies did not evaluate the possible cytotoxic effects of these solutions. This should also be taken into account before choosing the most appropriate antiseptic solution

Aim. Candida species are uncommon pathogens causing prosthetic joint infection (PJI). This study evaluated the surgical management and outcome of Candida PJI. Methods. Patients with EBJIS Definition confirmed PJI, due to Candida species, from 19 medical centres were assessed. Demographic, diagnostic, medical and surgical treatment and outcome data were collected. Results. 269 patients were recruited with follow-up for at least one year. Mean age was 70.2 years (+/- 12.4) with 10.8% being immunocompromised. The most common fungal species were C. albicans (55.8%), C. parapsilosis (29.4%), C. glabrata (7.8%) and C. tropicalis (5.6%). Co-infection with bacteria occurred in 138 (51.3%) cases. DAIR was performed in 96 (36.2%) cases, with 169 (63.8%) having implant exchange or removal (76 one-stage, 78 two-stage, 11 removal/Girdlestone arthroplasty, 2 amputation). Patient demographics and antifungal therapy were similar in all surgical groups. Overall, treatment was successful in 156 (58%) cases. Failure was more likely in older patients (>70 years; p=0.008) and those who had DAIR (OR 1.945; 1.156-3.279; p=0.004). Failure was less likely with C. parapsilosis infection compared to C. albicans (31.6% vs 48%; p=0.037). DAIR patients had more co-infection with bacteria (63.5% vs 47.4%; p=0.013) and more previous surgeries (median 4 vs 3; p=0.007), but multivariate analysis showed that these were not independent risk factors for failure. There was no difference in mortality between DAIR patients and those with other surgery (13.5% vs 17.7%; p=0.372). DAIR was successful in 45/96 (46.9%) cases compared to 110/169 (65.1%) cases with other surgery (p<0.004). Early DAIR (surgery performed <I month from implantation/infection onset) was not more effective than late DAIR (surgery performed after 1 month)(early DAIR 44.4% cure vs 63.9% cure in late DAIR; p=0.004). Two-stage revision was successful in 54/78 (69.2%), which was significantly better than DAIR (p=0.003). One-stage revision was successful in 51/76 (67.1%) patients; also significantly better than DAIR (p=0.002), but equivalent to two-stage revision (p=0.777). Conclusion. DAIR was successful in less than half of patients with Candida PJI. We could not identify any subgroup which might have better outcomes with this surgical option. Interestingly, almost 90% of our patients with Candida PJI had no immunocompromise. One or two-stage revision offer a better option, if possible, and do not increase mortality

Aim. In trauma surgery, the development of biomaterial-associated infections (BAI) is one of the most common complications affecting trauma patients, requiring prolonged hospitalization and the intensive use of antibiotics. Following the attachment of bacteria on the surface of the biomaterial, the biofilm-forming bacteria could initiate a chronic implant-related infection. Despite the use of conventional local and systemic antibiotic therapies, persistent biofilms involve various resistance mechanisms that contribute to therapeutic failures. The development of in vivo chronic BAI models to optimize antibiofilm treatments is a major challenge. Indeed, the biofilm pathogenicity and the host response need to be finely regulated, and compatible with the animal lifestyle. Previously, a Galleria mellonella larvae model for the formation of an early-stage biofilm on the surface of a Kirschner (K)-wire was established. In the present study, two models of mature biofilm using clinical Staphylococcus aureus strains were assessed: one related to contaminated K-wires (in vitro biofilm maturation) and the second to hematogenous infections (in vivo biofilm maturation). Rifampicin was used as a standard drug for antibiofilm treatment. Method. In the first model, biofilms were formed following an incubation period (up to 7 days) in the CDC Biofilm Reactor (CBR, BioSurface Technologies). Then, after implantation of the pre-incubated K-wire in the larvae, rifampicin (80 mg/kg) was injected and the survival of the larvae was monitored. In the second model, biofilm formation was achieved after an incubation period (up to 7 days) inside the larvae and then, after removing the K-wires from the host, in vitro rifampicin susceptibility assays were performed (according to EUCAST). Results. The first model indicate that in vitro biofilm maturation affects the bacterial pathogenicity in the host, depending on the S. aureus strain used. Furthermore, the more the biofilm is matured, the more the rifampicin treatment efficiency is compromised. The second model shows that, despite the fast in vivo biofilm formation in the host, the number of bacteria, either attached to the surface of the K-wire surface or in surrounding tissue of the larvae, was not increased over time. Conclusions. Altogether, these results allow the establishment of biofilm models using G. mellonella larvae in order to understand the impact of biofilm maturation on both the bacterial pathogenicity and the efficiency of antibiofilm treatments

Aim. dalbavancin, a lipo-glycopeptide antibiotic effective against Gram-positive bacteria (including methicillin-resistant Staphylococcus aureus), allows extended dosing interval due to its peculiar pharmacokinetics. Despite being registered for treatment of acute skin infections, off-label use has shown promise in various settings, particularly in osteo-articular infections. This study aims to assess dalbavancin's pharmacological efficacy and its safety and clinical success in patients treated according to personalized schedules guided by Therapeutic Drug Monitoring (TDM), particularly in long-term therapies. Methods. non-interventional, retrospective, single-center pharmacological study. We included adult patients with at least one dalbavancin TDM determination from July 1, 2022 to February 1, 2024 and treated with outpatient parenteral antimicrobial therapy. We recorded dalbavancin trough concentration (Cmin) and its peak concentration (Cmax) and employed log-linear regression models to predict the timing of dalbavancin dosing, aiming to sustain Cmin levels above 4 or 8.04 mg/L, according to recent literature. Data regarding index infections, patients’ characteristics, outcomes, and adverse events were also collected. Results. we included 32 patients, whose clinical and microbiological characteristics are depicted in Table 1. Regarding the primary outcome, 132/134 (98.5%) trough concentration was >4 mg/L, while 112/134 (83.6%) was >8.04 mg/L. For the secondary outcomes, 2/32 patients experienced an adverse event correlated to dalbavancin: (i) exanthema one week after the start of therapy and (ii) exanthema, conjunctivitis, angioedema, and nausea one month after the start of therapy. Moreover, we observed 4/32 clinical unsuccess (one failure during treatment, one relapse after the end of therapy, one switch to another antibiotic, and one isolation of non-susceptible microorganism). Conclusions. a TDM-based approach with the use of a log-linear regression model allows a more precise timing of dalbavancin administration by maintaining sufficient concentration of circulating drugs. This approach is promising for infections requiring a long-term treatment, such as orthopedic infection where source control is not possible. For any tables or figures, please contact the authors directly

Aim. Chemical debridement is a fundamental step during Periprosthetic joint infection (PJI) surgery. Antiseptic solutions are commonly used, but evidence on the optimal antiseptic, concentration, and irrigation time is lacking. The aim of this study is to analyze and compare the anti-biofilm capacity of povidone iodine, H. 2. 0. 2. , acetic acid and Bactisure™ after different exposure times, as well as their combinations. Method. Surgical steel discs inoculated with methicillin susceptible (MSSA) and resistant S. aureus (MRSA), P. aeruginosa, and S. epidermidis were exposed to the following antiseptic solutions: 0.3% (PI0.3) and 10% povidone iodine (PI10), H. 2. 0. 2. , 3% Acetic acid (AA3) and Bactisure™. Combinations included AA3, H. 2. 0. 2. , and PI10 in various orders. Exposure time for the antiseptics solutions was 1, 3 and 5 minutes, while combinations had a 9-minute total exposure, 3 minutes per antiseptic sequentially. All experiments were performed in triplicate and with a sterile saline control. nThe reduction in colony-forming units (CFU) was measured after sonication, and biofilm structure was analyzed via scanning electron microscopy. Results. PI showed the highest antibiofilm activity. PI0.3 eradicated bacteria on the discs after 3 and 5 minutes of exposure, but only achieved a 77.1% reduction after 1 minute. After PI10 treatment, we did not recover any bacteria regardless of exposure time. H. 2. 0. 2. , AA3, and Bactisure™ reached a significantly lower bacterial decrease at all exposure times compared to PI0.3 and PI10. AA3 was less effective against MSSA and S. epidermidis. H. 2. 0. 2. showed less activity against MRSA than PI0.3, PI10, and Bactisure™. Combinations of antiseptics starting with AA3 showed the best results in terms of CFU reduction and cell viability. Conclusions. We propose a sequential combination of AA3 + H. 2. 0. 2. + PI10 with an exposure time of 9 minutes for the chemical debridement in PJI surgery. First, AA3 performs debridement and disruption of the biofilm. Then, H. 2. 0. 2. has a bactericidal effect and increases the porosity of the cell wall, and PI10 has a final bactericidal effect. If combinations are unavailable, PI is a cost-effective alternative

Aim. Dalbavancin is a lipoglycopeptide with a broad antimicrobial spectrum against Gram-positive bacteria and effect against microorganisms in biofilm in vitro. Its pharmacokinetic properties, with an exceptionally long half-life of approximately 300 hours, allow for simplified administration that may be of value in the long-term treatment of bone and joint infections, such as prosthetic joint infections (PJIs). Several case reports and case series with “off-lable” treatment with dalbavancin of PJIs exist, but the optimal dosing regimen remains to be defined. Therapeutic drug monitoring (TDM) is recommended for treatment with >2 doses of dalbavancin. In the absence of TDM, the Swedish national guidelines for bone and joint infections (2023, . www.infektion.net. ) recommends a loading dose of dalbavancin 1,500 mg on day 1 and 1,500 mg on days 8 – 14, after which from day 28 1,000 mg is given biweekly or 500 mg every week. The aim of the present study was to determine trough levels of dalbavancin in patients with long-term treatment of PJIs according to the national guidelines. Method. Twelve patients with PJI were treated with at least 6 doses of dalbavancin, of which the first two doses were 1500 mg and the following doses were 1000 every second week, and prospectively sampled biweekly for determination of serum concentrations (trough levels) of dalbavancin which was measured by liquid chromatography coupled to electrospray tandem mass spectrometry (LC-MS/MS). The renal function was also examined. Results. The median serum concentration 14 days after the first dose of dalbavancin 1500 mg was 36.3 mg/L (range 6.6 – 62.4 mg/L). The median value 14 days after the second dose of 1500 mg (day 27 – 28) was 48.2 mg/L (range 12.2 – 77.3 mg/L). The trough value after the last dose of a total of 6 – 7 doses was as median 43.1 mg/L (range 26.2 – 97.5 mg/L). Three patients showed a tendency towards successive accumulation of dalbavancin during treatment. None of the patients, including those three with increasing through levels during treatment, showed any significant alteration in creatinine nor glomerular filtration rate. Conclusions. TDM during long-term treatment with dalbavancin is recommended to avoid the risk of accumulation and unnecessarily high trough values. With TDM, the dosing interval can be extended in several cases. In addition, with the support of TDM, subtherapeutic serum concentrations, with the risk of developing resistance, can be avoided

Introduction. Bone and joint infection (BJI) is often characterized by severe inflammation and progressive bone destruction. Osteocytes are the most numerous and long-lived bone cell type, and therefore represent a potentially important long-term reservoir of bacterial infection. Staphylococcus aureus is known to establish stable intracellular osteocytic infections, however, little is known about the less virulent yet second most prevalent BJI pathogen, S. epidermidis, associated with late-diagnosed, chronic BJI. Thus, this study sought to establish an in vitro model to study the infection characteristics of S. epidermidis in human osteocyte-like cells. Methods. SaOS2 cells (1 ×10. 4. cells/cm. 2. ) were grown to confluence either without differentiation, representing an osteoblast-like (OB) state (SaOS2-OB) or differentiated to an osteocyte-like stage (SaOS2-OY), using established methods. Four S. epidermidis strains used (ATCC-12228, ATCC-14990, ATCC-35984 and a clinical osteomyelitis strain RAH-SE1) were tested to be Lysostaphin-resistant, necessitating antibiotic (Levofloxacin) control of extracellular bacteria. Infection of host cells (OB or OY) was tested at three multiplicities of infection (MOI: 10, 100 and 1000). Extracellular bacteria were controlled by overnight incubation at a 10X minimum inhibitory concentration (MIC) of Levofloxacin and thereafter at 1XMIC. At each time point (days 1, 3, 5) viable intra- and extracellular bacteria were quantified. Result. All strains displayed similar intracellular infection and persistence capabilities in SaOS2-OB and SaOS2-OY. Independent of MOI, intracellular bacteria in SaOS2-OB decreased over time, becoming non-culturable by day 5. In contrast, SaOs2-OY displayed enhanced intracellular bacterial persistence at each time point. In the presence of increased Levofloxacin concentration (10XMIC), S. epidermidis could persist intracellularly for at least 14 days. Conclusion. This study showed for the first time that S. epidermidis can infect human osteocytes and persist intracellularly. Additionally, even a 10xMIC antibiotic concentration failed to eradicate intracellular bacteria, suggesting that persistence within osteocytes could contribute to treatment failure and establishment of chronic BJI

Aims. The mechanism by which synovial fluid (SF) kills bacteria has not yet been elucidated, and a better understanding is needed. We sought to analyze the antimicrobial properties of exogenous copper in human SF against Staphylococcus aureus. Methods. We performed in vitro growth and viability assays to determine the capability of S. aureus to survive in SF with the addition of 10 µM of copper. We determined the minimum bactericidal concentration of copper (MBC-Cu) and evaluated its sensitivity to killing, comparing wild type (WT) and CopAZB-deficient USA300 strains. Results. UAMS-1 demonstrated a greater sensitivity to SF compared to USA300 WT at 12 hours (p = 0.001) and 24 hours (p = 0.027). UAMS-1 died in statistically significant quantities at 24 hours (p = 0.017), and USA300 WT survived at 24 hours. UAMS-1 was more susceptible to the addition of copper at four (p = 0.001), 12 (p = 0.005), and 24 hours (p = 0.006). We confirmed a high sensitivity to killing with the addition of exogenous copper on both strains at four (p = 0.011), 12 (p = 0.011), and 24 hours (p = 0.011). WT and CopAZB-deficient USA300 strains significantly died in SF, demonstrating a MBC-Cu of 50 µM against USA300 WT (p = 0.011). Conclusion. SF has antimicrobial properties against S. aureus, and UAMS-1 was more sensitive than USA300 WT. Adding 10 µM of copper was highly toxic, confirming its bactericidal effect. We found CopAZB proteins to be involved in copper effluxion by demonstrating the high sensitivity of mutant strains to lower copper concentrations. Thus, we propose CopAZB proteins as potential targets and use exogenous copper as a treatment alternative against S. aureus. Cite this article: Bone Joint Res 2024;13(11):632–646

Aims. The efficacy of saline irrigation for treatment of implant-associated infections is limited in the presence of porous metallic implants. This study evaluated the therapeutic efficacy of antibiotic doped bioceramic (vancomycin/tobramycin-doped polyvinyl alcohol composite (PVA-VAN/TOB-P)) after saline wash in a mouse infection model implanted with titanium cylinders. Methods. Air pouches created in female BalBc mice by subcutaneous injection of air. In the first of two independent studies, pouches were implanted with titanium cylinders (400, 700, and 100 µm pore sizes) and inoculated with Staphylococcus aureus (1 × 10. 3. or 1 × 10. 6. colony-forming units (CFU)/pouch) to establish infection and biofilm formation. Mice were killed after one week for microbiological analysis. In the second study, pouches were implanted with 400 µm titanium cylinders and inoculated with S. aureus (1 × 10. 3. or 1 × 10. 6. CFU/pouch). Four groups were tested: 1) no bacteria; 2) bacteria without saline wash; 3) saline wash only; and 4) saline wash plus PVA-VAN/TOB-P. After seven days, the pouches were opened and washed with saline alone, or had an additional injection of PVA-VAN/TOB-P. Mice were killed 14 days after pouch wash. Results. The first part of the study showed that low-grade infection was more significant in 400 µm cylinders than cylinders with larger pore sizes (p < 0.05). The second part of the study showed that saline wash alone was ineffective in eradicating both low- and high-grade infections. Saline plus PVA-VAN/TOB-P eradicated the titanium cylinder-associated infections, as manifested by negative cultures of the washouts and supported by scanning electron microscopy and histology. Conclusion. Porous titanium cylinders were vulnerable to bacterial infection and biofilm formation that could not be treated by saline irrigation alone. Application of PVA-VAN/TOB-P directly into the surgical site alone or after saline wash represents a feasible approach for prevention and/or treatment of porous implant-related infections. Cite this article: Bone Joint Res 2024;13(11):622–631